Roland Jakob-Roetne

Generation of vascular endothelial and smooth muscle cells from human pluripotent stem cells.

The use of human pluripotent stem cells for in vitro disease modelling and clinical applications requires protocols that convert these cells into relevant adult cell types. Here, we report the rapid and efficient differentiation of human pluripotent stem cells into vascular endothelial and smooth muscle cells. We found that GSK3 inhibition and BMP4 treatment rapidly committed pluripotent cells to a mesodermal fate and subsequent exposure to VEGF-A or PDGF-BB resulted in the differentiation of either endothelial or vascular smooth muscle cells, respectively. Both protocols produced mature cells with efficiencies exceeding 80% within six days. On purification to 99% via surface markers, endothelial cells maintained their identity, as assessed by marker gene expression, and showed relevant in vitro and in vivo functionality. Global transcriptional and metabolomic analyses confirmed that the cells closely resembled their in vivo counterparts. Our results suggest that these cells could be used to faithfully model human disease.

Structure-based design, synthesis, and in vitro evaluation of bisubstrate inhibitors for catechol O-methyltransferase (COMT).

The enzyme catechol O-methyltransferase (COMT) catalyzes the Me group transfer from the cofactor S-adenosylmethionine (SAM) to the hydroxy group of catechol substrates. Potential bisubstrate inhibitors of COMT were developed by structure-based design and synthesized. The compounds were tested for in vitro inhibitory activity against COMT obtained from rat liver, and the inhibition kinetics were examined with regard to the binding sites of cofactor and substrate. One of the designed molecules was found to be a bisubstrate inhibitor of COMT with an IC50 = 2 microM. It exhibits competitive kinetics for the SAM and noncompetitive kinetics for the catechol binding site. Useful structure-activity relationships were established which provide important guidelines for the design of future generations of bisubstrate inhibitors of COMT.

Bisubstrate inhibitors for the enzyme catechol-O-methyltransferase (COMT): influence of inhibitor preorganisation and linker length between the two substrate moieties on binding affinity.

Inhibition of the enzyme catechol-O-methyltransferase (COMT) is an important approach in the treatment of Parkinsons disease. A series of new potent bisubstrate inhibitors for COMT, resulting from X-ray structure-based design and featuring adenosine and catechol moieties have been synthesised. Biological results show a large dependence of binding affinity on inhibitor preorganisation and the length of the linker between nucleoside and catechol moieties. The most potent bisubstrate inhibitor for COMT has an IC50 value of 9 nM. It exhibits competitive kinetics for the SAM and mixed inhibition kinetics for the catechol binding site. Its bisubstrate binding mode was confirmed by X-ray structure analysis of the ternary complex formed by the inhibitor, COMT and a Mg2+ ion.

X‐ray Crystal Structure of a Bisubstrate Inhibitor Bound to the Enzyme Catechol‐O‐methyltransferase: A Dramatic Effect of Inhibitor Preorganization on Binding Affinity

With an IC50 value of 9 nM, 1 is the most potent known disubstrate inhibitor for catechol-O-methyltransferase (COMT). Inhibition of COMT is of significant interest in the therapy of Parkinsonapos;s disease since it ensures that a larger percentage of orally administered L-dopa reaches-in the form of dopamine-its target in the brain. The X-ray crystal structure of a complex formed by COMT and 1 has been solved at 2.6-A resolution.

Molecular recognition at the active site of catechol-o-methyltransferase: energetically favorable replacement of a water molecule imported by a bisubstrate inhibitor.

Biologically active catechols, such as l-DOPA and the neurotransmitter dopamine, are inactivated by methylation. This reaction is catalyzed by the enzyme catechol-O-methyltransferase (COMT) in the presence of S-adenosylmethionine (SAM) and Mg ions. Small nitrocatechol-based inhibitors of COMT find application in the treatment of Parkinson disease by blocking unwanted methylation of the administered l-DOPA, thereby enhancing dopamine levels in the brain. 3] Recent studies have pointed towards additional therapeutic applications of COMT inhibition in other disorders of the central nervous system, such as schizophrenia and depression. We have developed a series of potent bisubstrate inhibitors for COMT which are competitive for both the catechol and the SAM binding sites. Based on the X-ray crystal structure of ligand 1 (IC50 = 9 nm) [7a] in a ternary complex with COMT and a Mg ion (PDB code: 1JR4), we started a detailed exploration of the molecular recognition properties of the entire active site of the enzyme. Importantly, we found that potentially hepatotoxic nitro groups, which are mandatory in catechol-based monosubstrate inhibitors, are not required for high-affinity bisubstrate inhibition. We substituted the nitro group in position 5 of 1 with appropriate lipophilic residues, such as the 4-fluorophenyl ring in 2 (IC50 = 31 nm), and found that the high, competitive inhibitory potency was maintained. Computer modeling studies suggested that the newly introduced lipophilic residue occupies a hydrophobic cleft near the surface of the enzyme. This initial proposal is validated here experimentally by X-ray crystallography. The crystal structure of 1 in a ternary complex with COMT and a Mg ion shows that the adenine moiety forms two hydrogen bonds, a moderately strong and a weak one (d(N···O): 3.0 and 3.4 , respectively), to a water molecule

Bisubstrate Inhibitors of the Enzyme Catechol O-Methyltransferase (COMT): Efficient Inhibition Despite the Lack of a Nitro Group

Catechol O-methyltransferase (COMT) catalyzes the O-methylation of catechols by S-adenosylmethionine (SAM) in the presence of Mg ions. Inhibition of COMT offers a therapeutic handle to reduce catecholamine metabolism, therefore providing a valuable complement for the treatment of CNS (central nervous system) disorders, such as Parkinson’s disease and possibly schizophrenia. The most efficacious therapy for Parkinson’s disease uses l-Dopa. The introduction of COMT-inhibitors (tolcapone (Tasmar8) and entacapone (Comtan8)) as adjuncts to this treatment has resulted in considerable therapeutic improvement, helping to substantially prolong the efficacy of l-Dopa dosage by preventing its catabolism through O-methylation. On the other hand, in some cases, adverse effects of hepatotoxicity have been associated with the use of tolcapone. It has been hypothesized that the hepatotoxic effect may be related to the nitrocatechol core structure of the drug. Therefore, the preparation of COMT inhibitors lacking the nitro group might be of advantage. However, this group is regarded as a key element for tight and reversible binding to the substrate pocket in the active site. Substitution of the nitro group by weaker electron-withdrawing substituents drastically reduces the affinity of catechols to COMT. Furthermore, the electron-withdrawing effect of the nitro group is reflected by a

Molecular Recognition at the Active Site of Catechol-O-methyltransferase (COMT): Adenine Replacements in Bisubstrate Inhibitors

L-Dopa, the standard therapeutic for Parkinsons disease, is inactivated by the enzyme catechol-O-methyltransferase (COMT). COMT catalyzes the transfer of an activated methyl group from S-adenosylmethionine (SAM) to its catechol substrates, such as L-dopa, in the presence of magnesium ions. The molecular recognition properties of the SAM-binding site of COMT have been investigated only sparsely. Here, we explore this site by structural alterations of the adenine moiety of bisubstrate inhibitors. The molecular recognition of adenine is of special interest due to the great abundance and importance of this nucleobase in biological systems. Novel bisubstrate inhibitors with adenine replacements were developed by structure-based design and synthesized using a nucleosidation protocol introduced by Vorbrüggen and co-workers. Key interactions of the adenine moiety with COMT were measured with a radiochemical assay. Several bisubstrate inhibitors, most notably the adenine replacements thiopyridine, purine, N-methyladenine, and 6-methylpurine, displayed nanomolar IC(50) values (median inhibitory concentration) for COMT down to 6 nM. A series of six cocrystal structures of the bisubstrate inhibitors in ternary complexes with COMT and Mg(2+) confirm our predicted binding mode of the adenine replacements. The cocrystal structure of an inhibitor bearing no nucleobase can be regarded as an intermediate along the reaction coordinate of bisubstrate inhibitor binding to COMT. Our studies show that solvation varies with the type of adenine replacement, whereas among the adenine derivatives, the nitrogen atom at position 1 is essential for high affinity, while the exocyclic amino group is most efficiently substituted by a methyl group.

Bisubstrate Inhibitors of Catechol O-Methyltransferase (COMT): the Crucial Role of the Ribose Structural Unit for Inhibitor Binding Affinity

Inhibition of the enzyme catechol O‐methyltransferase offers a therapeutic handle to regulate the catabolism of catecholamine neurotransmitters, providing valuable assistance in the treatment of CNS disorders such as Parkinsons disease. A series of ribose‐modified bisubstrate inhibitors of COMT featuring 2′‐deoxy‐, 3′‐deoxy‐, 2′‐aminodeoxy‐3′‐deoxy‐, and 2′‐deoxy‐3′‐aminodeoxyribose‐derived central moieties and analogues containing the carbocyclic skeleton of the natural product aristeromycin were synthesized and evaluated to investigate the molecular recognition properties of the ribose binding site in the enzyme. Key synthetic intermediates in the ribose‐derived series were obtained by deoxygenative [1,2]‐hydride shift rearrangement of adenosine derivatives; highlights in the synthesis of carbocyclic aristeromycin analogues include a diastereoselective cyclopropanation step and nucleobase introduction with a modified Mitsunobu protocol. In vitro biological evaluation and kinetic studies revealed dramatic effects of the ribose modification on binding affinity: 3′‐deoxygenation of the ribose gave potent inhibitors (IC50 values in the nanomolar range), which stands in sharp contrast to the remarkable decrease in potency observed for 2′‐deoxy derivatives (IC50 values in the micromolar range). Aminodeoxy analogues were only weakly active, whereas the change of the tetrahydrofuran skeleton to a carbocycle unexpectedly led to a complete loss of biological activity. These results confirm that the ribose structural unit of the bisubstrate inhibitors of COMT is a key element of molecular recognition and that modifications thereof are delicate and may lead to surprises.

The dynamics of Aβ distribution after γ-secretase inhibitor treatment, as determined by experimental and modelling approaches in a wild type rat.

Inhibition of the enzyme(s) that produce the Amyloid beta (Aβ) peptide, namely BACE and γ-secretase, is considered an attractive target for Alzheimer’s disease therapy. However, the optimal pharmacokinetic–pharmacodynamic modelling method to describe the changes in Aβ levels after drug treatment is unclear. In this study, turnover models were employed to describe Aβ levels following treatment with the γ-secretase inhibitor RO5036450, in the wild type rat. Initially, Aβ level changes in the brain, cerebral spinal fluid (CSF) and plasma were modeled as separate biological compartments, which allowed the estimation of a compound IC50 and Aβ turnover. While the data were well described, the model did not take into consideration that the CSF pool of Aβ most likely originates from the brain via the CSF drainage pathway. Therefore, a separate model was carried out, with the assumption that CSF Aβ levels originated from the brain. The optimal model that described the data involved two brain Aβ 40 sub-compartments, one with a rapid turnover, from which CSF Aβ 40 is derived, and a second quasi-static pool of ~20%. Importantly, the estimated in vivo brain IC50 was in a good range of the in vitro IC50 (ratio, 1.4). In conclusion, the PK/PD models presented here are well suited for describing the temporal changes in Aβ levels that occur after treatment with an Aβ lowering drug, and identifying physiological parameters.